Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity.


The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics. Study holds ProTherm entries: 25489, 25490, 25491, 25492, 25493, 25494, 25495, 25496, 25497, 25498 Extra Details: conformational stability; thermal stability; ligand-binding affinity; linkage analysis; thiol protection

Submission Details

ID: joyYVDep3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Isom DG;Vardy E;Oas TG;Hellinga HW,Proc. Natl. Acad. Sci. U.S.A. (2010) Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity. PMID:20194783
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thermonuclease P00644 NUC_STAAU
99.3 Thermonuclease Q5HHM4 NUC_STAAC
99.1 Thermonuclease Q99VJ0 NUC_STAAM
99.1 Thermonuclease Q7A6P2 NUC_STAAN
99.3 Thermonuclease Q6GB41 NUC_STAAS
99.3 Thermonuclease Q8NXI6 NUC_STAAW
99.3 Thermonuclease Q6GIK1 NUC_STAAR
100.0 Ribose import binding protein RbsB P02925 RBSB_ECOLI
97.3 Ribose import binding protein RbsB P0A2C6 RBSB_SALTI
97.3 Ribose import binding protein RbsB P0A2C5 RBSB_SALTY